Figure 3. Partial colocalization of H_v1 and Nox2 in different types of human granulocytes.

(a) Detection of H_v1 (left-most column) and Nox2 (middle) in granulocytes. (Scale bars represent 5 µm). Pretreating neutrophils with DFP improved the detection of H_v1. Colocalization analysis was performed only in cells, in which above-threshold labeling (two times above background, see below) for both proteins could be detected. Colocalizing pixels are displayed as white dots in the most right column. Negligible Alexa Fluor® signals (considered as background) were detected with control primary antibodies (not shown). (b) Western blot analysis of the distribution of H_v1 and Nox2 between granule fractions of resting neutrophils after standard reducing PAGE. Ponceau stain confirmed that similar protein amount was loaded each lane (not shown). The different fractions are: azurophil (α), specific (β₁), gelatinase (β₂) granules and secretory vesicle together with plasma membrane (γ). Immunodetection of lactoferrin, gelatinase, CD14 and the heavy chain of myeloperoxidase (MPO_HC) demonstrates the purity of the membrane preparates [22].